Ocular Hypotensive Response in Nonhuman Primates of (8R

Article pubs.acs.org/jmc

Ocular Hypotensive Response in Nonhuman Primates of (8R)‑1[(2S)‑2-Aminopropyl]-8,9-dihydro‑7H‑pyrano[2,3‑g]indazol-8-ol a Selective 5‑HT2 Receptor Agonist Jesse A. May,*,† Najam A. Sharif,† Marsha A. McLaughlin,† Hwang-Hsing Chen,† Bryon S. Severns,† Curtis R. Kelly,† William F. Holt,† Richard Young,‡ Richard A. Glennon,‡ Mark R. Hellberg,† and Thomas R. Dean† †

Ophthalmology Discovery Research, Alcon Research, Ltd., 6201 South Freeway, Fort Worth, Texas 76134, United States Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Box 980540, Richmond, Virginia 23298, United States

‡

S Supporting Information *

ABSTRACT: Recently, it has been reported that 5-HT2 receptor agonists effectively reduce intraocular pressure (IOP) in a nonhuman primate model of glaucoma. Although 1-[(2S)-2-aminopropyl]indazol-6-ol (AL-34662) was shown to have good efficacy in this nonhuman primate model of ocular hypertension as well as a desirable physicochemical and permeability profile, subsequently identified cardiovascular side effects in multiple species precluded further clinical evaluation of this compound. Herein, we report selected structural modifications that resulted in the identification of (8R)-1-[(2S)-2-aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol8-ol (13), which displayed an acceptable profile to support advancement for further preclinical evaluation as a candidate for proof-of-concept studies in humans.

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INTRODUCTION Glaucoma is a leading cause of blindness, though presently there is no known cure for this disease. An elevation of intraocular pressure remains the primary indicator for projecting an increased probability of the onset of primary open angle glaucoma, an ocular neuropathy which can lead to the loss of vision.1,2 The reduction of this elevated intraocular pressure (IOP) has been demonstrated to be an effective treatment for preventing the progression of glaucoma.3 While a number of therapeutic agents are now available for the treatment of glaucoma through several different physiological mechanisms that lead to a decrease of IOP in an attempt to mitigate vision loss, there remains a significant portion of patients for whom these drugs are either contraindicated or not effective. There is, therefore, a critical medical need to identify alternate therapeutic approaches for efficiently decreasing the elevated IOP in patients with ocular hypertension in an attempt to maintain vision. This need is presently most urgent in view of an ever increasing percentage of the population entering the age-group cohort with the highest risk for the development of glaucoma. The presence of serotonin (5-hydroxytryptamine, 5-HT) in the eye is well established, and specific classes of the numerous receptors for this ubiquitous neurotransmitter have been identified in the ocular tissues of the anterior segment of the eye; several of these receptors have been suggested to have a significant role in maintaining normal IOP. The specific roles reported for the different 5-HT receptors based on animal studies has been somewhat contradictive and further has shown © XXXX American Chemical Society

an apparent species variation; hence, the influence of these 5HT receptors remains unclear.4 The expression of 5-HT2 receptor mRNA in human ciliary body, ciliary muscle, and trabecular meshwork cells has been noted, and furthermore, functional 5-HT2 receptors have been identified in the ocular tissues critical to modulating IOP such as the ciliary epithelium, ciliary muscle, and trabecular meshwork cells.5−8 The presence of functionally active 5-HT2 receptors in these tissues known to be involved in the control of IOP suggests that serotonin may have an important role in the regulation of IOP through these receptors. Activation of 5-HT2 receptors following the topical ocular administration of 5-HT2 receptor agonists has been shown to result in a significant reduction of IOP in a nonhuman primate (NHP) model of ocular hypertension.9 We have previously reported that a peripherally acting selective 5-HT2 receptor agonist 1-[(2S)-2-aminopropyl]indazol-6-ol (1, AL-34662, Figure 1) exhibited good efficacy in a NHP model of ocular hypertension.10 However, this compound was subsequently shown to be unacceptable for further consideration as a clinical proof-of-concept candidate based on undesirable cardiovascular issues observed during late stage preclinical in vivo pharmacology evaluations. This response was hypothesized to be due to the previously unidentified potent α1D receptor agonist activity of the molecule (vide infra). Therefore, identification of a compound lacking this activity was of interest. In view of the favorable Received: June 4, 2015

A

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caused by 3; this significant CNS liability precluded any further evaluation of compound 3 in monkeys.12 It is of interest to note that though the comparable tPSA values for 2 and 3 were suggestive of low CNS permeability for these compounds, the experimentally determined DC7.4 and Papp PD values more favorably predicted the outcome observed from the in vivo studies.11,12 Herein, we report selected structural modifications of 3 that were evaluated and resulted in the identification of a lead compound within this series, which displayed an acceptable profile to support advancement for further preclinical evaluation as a candidate for proof-of-concept studies in humans.

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CHEMISTRY Scheme 1 outlines the synthesis of epimers 13 and 14, initiated by the protection of 510 with t-butyldimethylchlorosilane to give 6 followed by debenzylation with Pd/C under hydrogen to give phenol 7, which upon subsequent bromination with Nbromosuccinimide in tetrahydrofuran gave the 7-bromo-6hydroxy indazole 8 in good yield; no additional bromination products were identified. Alkylation of 8 with epibromohydrin in the presence of potassium carbonate in acetone afforded epoxide 9, which was treated with magnesium and 1,2dibromoethane to give the 6-(1-bromo-2-hydroxy-propoxy) intermediate that was immediately treated with ethyl vinyl ether to give the O-protected compound 10. Metalation of 10 with nbutyllithium at −78 °C with subsequent cyclization, along with concomitant cleavage of the silyl ether protecting group, led to the formation of pyranoindazole 11. Treatment of 11 with methanesulfonic anhydride and then sodium azide in DMF gave the intermediate azide, which was deprotected to afford 12. The stereoisomers of compound 12 were separated by chiral HPLC to give 12a and 12b, hydrogenation of which gave the desired amines 13 and 14, respectively. An alternative asymmetric synthetic method for obtaining compound 12a is shown in Scheme 2. Alkylation of compound 7 with propargyl bromide in the presence of potassium carbonate in acetone gave the 6-propargyloxyindazole 15, which readily underwent a

Figure 1. Structures.

ocular antihypertensive efficacy and lack of CNS effects observed for the pyrano[3,2-e]indole compound 2 (AL37350A, Figure 1),11 but which lacked acceptable solution stability due to the indole moiety, it was of interest to explore the efficacy of the corresponding pyrano[2,3-g]indazole compound 3 (AL-38022A, Figure 1), which would be expected to have an increased solution stability comparable to that of the indazole analogue 1. A comparison between these two fused pyrano-heterocyclic analogues showed that 3 did indeed display a greatly increased solution stability: estimated half-life (t1/2) as an aqueous solution (pH 7.4) of 15.1 years (25 °C).12 The comparable calculated polar surface areas for these two compounds (Table 1) suggested that 3 might also lack unfavorable CNS effects. However, there was concern that permeability into the CNS might be problematic since the distribution coefficient for 3 (Table 1) was about 15-fold higher than that for 2, and the MDCK/MDR cell permeability Papp‑PD for 3 was 9-fold higher than that for 2; and furthermore, 2 was a substrate for the P-gp efflux system, while 3 was not a substrate (Table 1). It was subsequently established during an acute rabbit safety study that extremely severe CNS effects were

Table 1. Physiochemical and Permeability Data for Compounds and Topically Dosed Ocular Drugs cmpds (4, R = ) 2 3 13 20 25 28 32a 32b 32c 32d

-OH -NH2 -OCH2CH2NH2 -OCH2CH2OH -OCH2CONHCH3 -OCH2CONH-(CH2)2OCH3 -OCH2CONH-(CH2)3NH2 -OCH2CO-[N(CH2CH2)2N](pyridin-2-yl) timolol betaxolol dorzolamide brinzolamide apraclonidine brimonidine pilocarpine

tPSAa (Å2)

DC7.4b

Papp PD, nm/sc[P-gp substrate, efflux ratio]

51.0 53.1 73.3 79.1 88.3 82.5 91.4 101 117 96.0

0.281 4.39e 0.349 0.0 0.0 0.223 0.294 0.312 < 0.10 < 0.10

18.5 [yes, 9.3] 164 [no]e 5.4 [yes, 5.8] 2.5 [no, 2.8] 1.6 [no, 1.3] 1.6 [yes, 22] 5.0 [yes, 5.0] 4.3 [yes, 7.0] < 6.0 [no] 2.8 [yes, 22]

108 50.7 151 164 62.4 62.2 44.1

1.16 6.34 1.72 6.56 0.018 1.53 1.39

33.9 [yes, 26.2] 202 [yes, 6.0] 4.0 [yes, 7.8] 11.7 [yes, 68] 3.8 [no] 30.9 [no] 36.8 [no]

rabbit cornead Papp nm/s (±sd)

rabbit conjunctivadPapp nm/s (±sd)

451 ± 65 53 ± 6 11 ± 2 13 ± 2 44 ± 6 20 ± 6

298 ± 37 87 ± 59 38 ± 5 69 ± 8 242 ± 62 141 ± 55

45 ± 7

72 ± 34

242 ± 3 232 ± 10 9.0 ± 9 4.4 ± 1.0 21 ± 7 238 ± 4 189 ± 9

94.2 ± 22 61.1 ± 3 15.5 ± 10 37.1 ± 27 29 ± 13 79.2 ± 37 248 ± 76

a

Calculated with ACD/logP, v.12.01. bExperimental values. cMDCK(MDR) cell permeability, by Absorption Systems, L.P. dEx vivo experiments by Absorption Systems, L.P. eData are from ref 12. B

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Scheme 1a

a Reagents and conditions: *Ref 10. (a) t-Butyldimethylchlorosilane, imidazole, THF; (b) H2, Pd/C, MeOH; (c) NBS, THF; (d) epibromohydrin, K2CO3, acetone; (e) (1) Mg, 1,2-dibromoethane, THF; (2) ethyl vinyl ether, TsOH, DCM; (f) (1) n-butyllithium, THF, −78 °C; (2) TBAF, THF; (g) (1) Ms2O, TEA, THF; (2) NaN3, DMF; (3) HCl; (h) chiral HPLC separation; (i) H2, Pd/C, MeOH.

analogue 25 from 17a is outlined in Scheme 3: sequential Oalkylation (21), removal of the silyl protecting group (22), and reduction of the ester gave the diol 23, which was converted to the desired diazide (24) and then was reduced to the targeted 2-aminoethoxy compound 25. Conversion of the R-hydroxyl group of ester 22 to the S-azide (26) in the manner described above followed by treatment with LAH, which reduced both the ester and the azide, gave the desired crude amino alcohol; however, to expedite purification of this compound the amine was protected with a carbobenzyloxy group (27). Following purification, reductive cleavage of the protecting group provided the desired compound 28 (Scheme 3). The amide analogues 32a−d were synthesized in four steps from the chiral alcohol 12a as illustrated in Scheme 4. This sequence began with O-alkylation using sodium hydride and isopropyl 2bromoacetate to afford 29 followed by transesterification with

Claisen rearrangement−cyclization when heated in mesitylene (200 °C) to give the C7 cyclization product pyrano[2,3g]indazole 16, in good yield; the C5 cyclization product (pyrano[2,3-f ]indazole) was not detected. Hydroboration of compound 16 with 9-BBN followed by treatment with hydrogen peroxide gave a mixture of the 8-hydroxy epimers 17a (R-C8-OH) and 17b (S-C8-OH) in a 9:1 ratio. The pure major isomer 17a was obtained by column chromatography. Protection of 17a with ethyl vinyl ether followed by treatment with tetra-n-butylammonium fluoride gave alcohol 18, which was converted to compound 12a using the same procedure as that earlier described for the preparation of compound 12 from 11 (Scheme 1). Synthesis of the 8-(R)-amino analogue of compound 13 was accomplished by the conversion of 12b to the diazide 19, and subsequent reduction provided the diamine 20 (Scheme 3). Preparation of the 8-(R)-2-aminoethoxy C

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Scheme 2a

Reagents and conditions: (a) propargyl bromide, K2CO3, acetone; (b) mesitylene, 200 °C; (c) (1) 9-BBN, THF; (2) H2O2, MeOH; (3) column chromatography; (d) (1) pyridine-TsOH, ethyl vinyl ether; (2) TBAF, THF; (e) (1) Ms2O, TEA, THF; (2) NaN3, DMF; (3) TsOH, MeOH; (4) TEA.

a

Scheme 3a

a

Reagents and conditions: (a) (1) Ms2O, TEA, THF; (2) NaN3, DMF; (b) H2, Pd/C, MeOH; (c) NaH, t-butylbromoacetate, DMF; (d) TBAF, THF; (e) LAH, THF; (f) (1) LAH, THF; (2) CbzCl, NaHCO3.

D

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Scheme 4a

substituent at C8 is well tolerated, and there does not appear to be a preferred stereochemistry for the substituent, at least for the hydroxyl group, since 13 and 14 have essentially the same functional response at the receptors; however, this substitution does result in a modest overall decrease in affinity at the 5-HT2 receptors as well as a decrease in both functional potency and efficacy at the 5-HT2 receptors relative to the unsubstituted compound 3 in the in vitro assays as listed in Table 2. The affinity of compounds at the three 5-HT2 receptor subtypes was generally of the order 5-HT2C ≥ 5-HT2A > 5-HT2B, and the functional response at the receptor subtypes displayed a similar preference; however, none of these compounds demonstrated a high level of selectivity for a single receptor subtype. The binding affinity data showed 32a to have only a marginal preference for 5-HT2C vs 5-HT2B (15-fold), which was not observed in the functional assay. Compound 13 and 28 showed a slight preference for 5-HT2C vs 5-HT2B (16-fold and 15-fold, respectively), in the functional assay; but only an equally modest preference for 5-HT2A or 5-HT2C vs 5-HT2B (10-fold and 15-fold, respectively) was observed in the functional assay for 32b. Compound 32d showed only a nominal preference for 5-HT2A/C vs 5-HT2B (≤10-fold) in this assay. The potent agonist response observed for 32d suggests that a region within the receptor has been identified that is capable of accommodating considerable steric bulk, indicating that perhaps the pyridyl-piperazinyl moiety reaches beyond the transmembrane domain of the receptor. Identification of such a location within the 5-HT2 receptor might find utility for subsequent inclusion of a suitable marker functionality within this framework to assist in exploring the role of ligand−receptor interactions at the molecular level. The possibility of compounds entering the CNS was further estimated by assessing their permeability of MDCK/MDR cells, a well-documented model of CNS penetration.13,14 Compounds of interest were those that had passive diffusion (Papp PD) of 30 μM). In Vivo Studies with the Lasered Cynomolgus Monkey Model of Chronic Ocular Hypertension. In this model, a test compound that decreases IOP in the lasered eye by more than 20% relative to the baseline pressure is considered to have a biologically significant response; however, a compound that decreases IOP by more than 25% is of particular interest as a potential candidate for further preclinical evaluation. As noted above, compounds of interest for evaluation in the monkey model initially undergo evaluation in an acute topical ocular rabbit safety study to assess suitability for in vivo testing. This preliminary safety study showed that compounds 13, 14, 20, and 28 had no significant adverse effects which would preclude their evaluation in ocular hypertensive monkeys; a summary of these studies (300 μg dose) is provided in Table 3. Compound 13, which provided an IOP reduction of greater than 25% at both the 3 and 6 h reading was the most effective of these compounds. Compound 14, the C8 epimer of 13, was less effective than 13 reducing IOP by 25% only at the 6 h time point and showing a modest reduction of 17% at the 3 h reading. Neither compound 20 nor 28 achieved the desired IOP reduction of 25% at any time point. As noted in Table 3, the IOP reduction of 13 showed a good dose−response relationship when further evaluated at 100 μg and 30 μg doses.

Table 3. Effect of Selected Compounds on IOP in Hypertensive Eyes of Conscious Cynomolgus Monkeys postdose IOP reduction, %b,c a

cmpd

dose (μg)

baseline IOP (mmHg)b

1h

13

30

33.9 (3.63)

0.9 (2.16)

100

36.9 (2.14)

9.2 (3.4)

300

35.1 (2.64)

7.6 (2.91)

14

300

34.3 (2.45)

7.9 (4.06)

20

300

36.0 (2.37)

7.4 (4.06)

28

300

38.5 (2.73)

7.6 (4.44)

3h 14.4f (3.65) 17.2e (2.3) 25.8e (3.53) 17.3e (3.87) 15.3d (3.23) 19.1e (4.40)

6h 19.9f (6.45) 25.7e (4.4) 30.2e (4.48) 25.1e (4.76) 20.8e (5.43) 19.8f (7.72)

a

Phosphate buffered saline, pH 7.4. bValue (SEM). cThe vehicle control group conducted with each study did not exceed the inherent model IOP variability of ±15%. dp < 0.001. ep < 0.01. fp < 0.5.

The 300 μg (1%) dose appears to be at the top of the dose− response curve since increasing the dose above this level (i.e., 2%, 4%) did not result in additional peak IOP reduction. On the basis of its overall favorable profile, compound 13 was selected as the preferred compound for further evaluation as described below. When compound 13 was dosed by topical ocular administration in the normotensive (nonlasered) eye of the monkeys, this treatment did not show significantly altered IOP in either the dosed normal eye or the contralateral lasered eye (Table 4). Also, compound 13 had no effect on the pupil since Table 4. IOP Response Following Topical Ocular Administration of 13 to the Normal Eye of Conscious Cynomolgus Monkeys postdose IOP reduction, %b,c a

dose (μg)

baseline IOP (mmHg)b

1h

3h

6h

300, OSd 0, ODe

19.3 (1.31) 30.9 (1.78)

2.7 (2.77) 2.4 (2.84)

9.3 (4.09) 5.7 (2.50)

10.5 (3.79) 10.6 (4.07)

a

Phosphate buffered saline, pH 7.4. bValue (SEM). cThe vehicle control group conducted with each study did not exceed inherent model IOP variability of ±15%. dNormal, normotensive treated left eye OS. eLasered (hypertensive, OD) right eye contralateral to treatment.

there was no mydriasis or miosis observed in the treated left eye, which had not undergone laser trabeculoplasty. Thus, the IOP lowering action of 13 appears to be locally mediated, rather than through a centrally or systemically mediated effect. It is of interest to note that compound 13 did not demonstrate any significant efficacy for IOP reduction in Dutch-belted rabbits that naturally have elevated IOPs compared to that of other strains of rabbit nor in normotensive cats (300 μg, mixed breed) following topical ocular administration of doses that were effective when applied to the hypertensive eye of monkeys. However, such species differences following topical ocular administration are not unprecedented; for example, neither rabbit nor cat IOP is modulated by prostaglandin FP-receptor agonists, while FPreceptor agonists are effective at reducing IOP in nonhuman primates as well as humans. Further, a reduction in IOP is F

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Table 5. Duration of IOP Reduction in Hypertensive Eyes of Conscious Cynomolgus Monkeys: Comparison between 13 and 1 postdose IOP reduction, %b,c a

dose (μg)

baseline IOP (mmHg)

300 300

34.6 (2.86) 35.4 (2.4)

13 1

b

1h

3h

6h

10 h

12 h

2/12 hd

5.5 (4.63) 7.4 (3.3)

18.4f (4.29) 18.8g (6.1)

22.5f (4.72) 20.7f (3.9)

25.9e (4.85) 21.5f (3.0)

27.9e (5.48) 17.8g (6.8)

26.7e (5.66) 15.3 (7.0)

a

Phosphate buffered saline, pH 7.4. bValue (SEM). cThe vehicle control group conducted with each study did not exceed inherent model IOP variability of ±15%. d2/12 indicates 12 h after second dose, 24 h after first dose. ep < 0.001. fp < 0.01. gp < 0.05.

Table 6. Duration of IOP Reduction in Hypertensive Eyes of Conscious Cynomolgus Monkeys: Exploratory Sustained Release Formulations of 13 postdose IOP reduction, %b,c a

b

dose (μg)

baseline IOP (mmHg)

1h

3h

6h

10 h

12 h

24 h

300d 300e 75f

39.9 (3.90) 36.8 (2.87) 39.6 (3.34)

13.4 (4.83) 11.8 (3.81) 10.4 (3.59)

28.1h (5.82) 29.8g (2.80) 23.2i (4.42)

34.4g (3.76) 28.0g (3.94) 23.9i (4.84)

39.6g (3.80)

32.5g (5.28)

33.7h (5.28)

29.1h (5.70)

36.6g (4.11) 27.7g (3.91) 24.4i (4.19)

a

b

c

Phosphate buffered saline, pH 7.4. Value (SEM). The vehicle control group conducted with each study did not exceed the inherent model IOP variability of ±15%. d0.5% Amberlite, QD. e0.1% Amberlite/0.001% DDP, QD. f0.6% xanthan gum, QD. gp < 0.001. hp < 0.01. ip < 0.05.

Table 7. Effect of Bromfenac on Lowering of IOP Induced by 13 in Ocular Hypertensive Cynomolgus Monkeys postdose IOP reduction, hypertensive eye (OD lasered), %b,c d

bromfenac /13 13

dosea (μg)

baseline IOP (mmHg)b

1h

3h

6h

120/150 150

44.6 (2.05) 41.8 (1.38)

13.1 (2.74)e 11.3 (2.16)e

25.0 (3.45)e 24.7 (2.79)e

28.9 (3.25)e 26.0 (3.45)e

a Phosphate buffered saline, pH 7.4. bValue (SEM). cThe vehicle control group conducted with each study did not exceed the inherent model IOP variability of ±15%. dBromfenac administered as one 30 μL drop, topical, and ocular every 15 min for 4 doses, then baseline IOP measured, waited for 10 min, and then dosed with 13; crossover with 1 week wash-out, n = 17. ep < 0.001.

to determine if the release of endogenous prostaglandins might be involved, at least in part, in the lowering of IOP observed with 13 by evaluating what effect a cyclooxygenase inhibitor might have on the IOP reduction observed for 13 in monkeys. Treatment of the hypertensive (lasered) eye of monkeys with 13, following pretreatment with bromfenac, a cyclooxygenase inhibitor well-known21 to readily traverse the cornea following topical ocular administration, provided a statistically significant reduction of IOP compared to the baseline value at each reading time point: 1 h (13.1%), 3 h (25.0%), and 6 h (28.9%) (Table 7). For comparison, the response for animals similarly dosed only with compound 13, but pretreated with the vehicle, also provided a statistically significant reduction of IOP at each reading time point: 1 h (11.3%), 3 h (24.7%), and 6 h (26.0%) (Table 7). The mean IOP in vehicle pretreated eyes was not significantly different from the mean IOP of bromfenac pretreated eyes at any time after dosing. Thus, pretreatment with bromfenac does not alter the IOP lowering effect of a 150 μg dose of 13 in hypertensive eyes in the monkey. These results suggest that the effect of 13 on IOP is not mediated by the synthesis and release of endogenous prostaglandins. Activation of the 5-HT2B receptors expressed on heart valve leaflets that leads to a valvular hyperplasia has now been established as a serious drug-induced heart disease observed for some therapeutic agents subsequent to chronic oral therapy.22−25 Most notable in this regard are the anorectic agent dfenfluramine (active metabolite, d-norfenfluramine) and the anti-Parkinsonism ergolines pergolide and cabergoline, also potent 5-HT2B receptor agonists.26 Hence, a compound with weak or no agonist activity at the 5-HT2B receptor would be most desirable. While the systemic concentration achieved in humans following topical ocular administration of 13 is not known at this point, this will need to be established, and there

observed in rabbits treated with prostaglandin DP-receptor agonists.17 Similarly, cabergoline lowers IOP in monkeys but not in Dutch-belted rabbits.18 The duration of the IOP effect of 13 in cynomolgus monkeys after a single a.m. dose (300 μg) followed by a subsequent single p.m. dose (300 μg) administered topically to the hypertensive right eye of the animals was evaluated; IOP reductions of 27.9% and 26.7% were observed 12 h after the morning and evening doses, respectively (Table 5). The IOP readings for compound 1, which was administered using the same protocol, showed a maximum reduction of 21.5% 10 h after the first dose, and the IOP was not maintained for 12 h after the second dose, suggesting a significant improvement in duration of action for 13 compared to 1. Further assessment of the duration of the IOP lowering effect of 13 in nonhuman primates was conducted by the topical administration of formulations of 13 that would be anticipated to significantly increase residence time of the compound on the ocular surface. As shown in Table 6, a low dose (75 μg) formulation gave good reduction of IOP at the 12 h reading (29.1%) but could not maintain the desired reduction to the 24 h time period. However, the two higher dose formulations (300 μg) demonstrated that a significant level of IOP reduction (36.6% and 27.7%) could be maintained in the treated hypertensive eye 24 h after administration of a single topical ocular dose. Agonist activation of the 5-HT2A receptor has been shown to differentially activate two signal transduction pathways: phospholipase C mediated inositol accumulation and phospholipase A2 mediated arachidonic acid release, where the stimulation of arachidonic acid release is independent of inositol accumulation.19,20 Since prostaglandins, i.e., FP and DP prostaglandin receptor agonists, have been shown to be efficacious17 in the treatment of glaucoma, it was of interest G

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5-HT2 receptor agonist with a comparable pharmacological profile to that of R-DOI, we suggest that 13 could also be mediating its IOP-lowering effects, at least in part, by the uveoscleral outflow pathway. However, since functional 5-HT2 receptors are also located on both human ciliary muscle and trabecular meshwork (TM) cells,6−8 it is possible that 13 might also promote aqueous humor outflow via the conventional TM pathway to lower IOP. In view of the well-known association of 5-HT2 receptor activation in the brain with hallucinations, it was necessary to assess the potential for these effects with 13; consequently, its discriminative stimulus effects were evaluated in a rat drug discrimination paradigm using DOM (1-(2,5-dimethoxy-4methylphenyl)-2-aminopropane) as the training drug.28,29 Unlike compound 3, which fully generalized to the DOM stimulus (ED50 = 0.05 mg/kg; Table 10), compound 13 did not

will be a need to carefully assess the posology for any eventual clinical candidate that might have even modest activity at this receptor to limit systemic exposure. However, the lack of selectivity among the 5-HT2 receptor subtypes for compound 13 is not considered detrimental at this stage; in fact, it remains to be determined whether or not a multireceptor subtype profile is a requirement for the robust IOP reduction observed. Evaluation of 13 in a large panel of relevant receptors, ionchannels, second messengers, enzymes, and transporters further demonstrates its selectivity for 5-HT2 receptors. Only low to weak affinity or functional response was noted for 13 at other 5-HT, adrenergic (alpha and beta subtypes), dopaminergic receptors and subtypes (Tables 8 and 9), or muscarinic, Table 8. Binding of 13 to Serotonergic Receptors and αAdrenergic Receptor Subtypes and Transmitter Uptake Systemsa receptor

IC50 (nM)a

5-HT1A 5-HT1D/1B 5-HT3 5-HT4 5-HT5A 5-HT6 5-HT uptake site α1A α1B β1 β2 D1 D2S D2L D4.2 norepinephrine uptake

687 2,100 >49,000 >100,000 >100,000 >100,000 >100,000 >100,000 >100,000 >78,000 42,000 >100,000 >100,000 >100,000 >100,000 >100,000

Table 10. Results of Drug Discrimination Studies for 13 and Selected Standard Compounds in DOM-Trained Rats and Doses for Human Response Where Known ED50 (mg/kg) cmpd DOM R-DOI 5-MeO− DMT 3 1 13

5-HT1A 5-HT7 α1D (rat aorta assay: agonism)c α2A (adenylyl cyclase assay: agonism) α2B (adenylyl cyclase assay: agonism) α2C (adenylyl cyclase assay: agonism)

> 10,000 >10,000 >30,000 >10,000 >10,000 >10,000

0.3c 0.2c 0.86c

0.2−0.6 0.1−0.3 0.50−1.45

1−5 1.5−3.0 (rac)e 5−10 (parenteral)

0.05c 32% @ 1f 12% @ 0.5f

0.03−0.10

generalize to the DOM stimulus, with the animals making only 12% of their responses on the DOM-appropriate lever at a dose of 0.5 mg/kg. Higher doses resulted in a disruption of behavior. Thus, the results of the rat drug discrimination study suggest that although to some extent 13 does appear to gain access to the CNS, it does not appear to elicit a DOM-like behavior in rats, suggesting a minimal likelihood for a hallucinogenic response in humans.

Table 9. Functional Response of 13 in Selected Serotonergic and Adrenergic Receptor Assays EC50 (nM)a

hallucinogenic dose, (mg, p.o.)a

Ref 38. bRef 28. cRef 12. dRef 29. eRef 39. fMaximum percentage DOM-appropriate responding at the highest responding-dose shown.

Data are an average of duplicate determinations performed at Alcon. The majority of these assays used an antagonist radioligand.

assay

0.44b 0.26d 1.22b

95% CL

assay

a

a

b

lit. value

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CONCLUSIONS Compound 13 has been shown to have an overall profile favorable for consideration as a candidate for further preclinical evaluation in support of a proof-of-concept study to evaluate the utility of 5-HT2 receptor agonists for the treatment of ocular hypertension and glaucoma in humans. Furthermore, it showed a long duration of IOP reduction in nonhuman primates; though it is difficult to predict duration of action across species, this observation is nonetheless quite encouraging for this new class of agents. Results from drug discrimination studies suggest that there are no apparent CNS issues associated with the potent 5-HT2A receptor agonist activity of this compound, which did not generalize to the DOM discriminative stimulus in rats. Additional studies will be required to determine the likelihood of any potential liability associated with 5-HT2B receptor agonist activity for such compounds when chronically dosed via a topical ocular route, compared to that observed following systemic delivery of therapeutics with this undesirable activity. In view of the extremely robust reduction of IOP observed for 13, further

Mean ± SEM and relative efficacy, Emax (%); all data were generated at Alcon except as noted. bInhibition of cAMP production. cAssay performed at MDS Pharma Services (Panlabs).

a

nicotinic, glutamatergic, GABAergic, opioid, sigma, and various ion-channels transporters and various peptidergic and growth factor/hormone receptors (Table S2, Supporting Information). Though the specific functional mechanism leading to the reduction of IOP by 5-HT2 receptor agonists remains to be determined, one possible operational mechanism of action for the lowering of IOP by these compounds has been suggested to be an increase in the flow of aqueous humor through the uveoscleral outflow pathway, as observed in monkeys treated with R-DOI, which is known to be a selective 5-HT2 receptor agonist.27 Since, as we have shown, compound 13 is a selective H

DOI: 10.1021/acs.jmedchem.5b00857 J. Med. Chem. XXXX, XXX, XXX−XXX

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103−105 °C; LC-MS (APCI) m/z 385, 387 [M + H]+; 1H NMR (CDCl3, 200 MHz) δ 7.90 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 5.87 (s, 1H), 4.93 (dd, J = 8.0, 14 Hz, 1H), 4.49 (m, 1H), 4.41 (m, 1H), 1.21 (d, J = 6.0 Hz, 3H), 0.73 (s, 9H), −0.15 (s, 3H), −0.46 (s, 3H). [(1R)-2-[7-Bromo-6-[3-bromo-2-(1-ethoxyethoxy)propoxy]indazol-1-yl]-1-methyl-ethoxy]-tert-butyl-dimethyl-silane (10). To a suspension of 8 (3.66 g, 9.51 mmol) and potassium carbonate powder (1.92 g, 1.46 mmol) in acetone (200 mL) was added epibromohydrin (1.32 mL, 15.1 mmol). The suspension was heated at reflux overnight, and the reaction was monitored by TLC. The suspension was cooled, filtered through diatomaceous earth, and evaporated to dryness. Column chromatography (silica, gradient, of ethyl acetate/hexane (2% to 10%) gave 9 ([(1R)-2-[7-bromo-6-(oxiran-2-ylmethoxy)indazol-1yl]-1-methyl-ethoxy]-tert-butyl-dimethyl-silane) as an oil (3.32 g, 79%): 1H NMR (CDCl3, 200 MHz): δ 7.92 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 4.95 (m, 1H), 4.61 (m, 1H), 4.40 (m, 1H), 4.17−4.11 (m, 2H), 3.43 (br s, 1H), 2.94−2.86 (m, 2H), 1.19 (d, J = 6.0 Hz, 3H), 0.73 (s, 9H), −0.14 (s, 3H), −0.41 (s, 3H). A mixture of magnesium (0.39 g, 16.1 mmol) and 1,2-dibromoethane (1.13 g, 6.02 mmol) in anhydrous THF (50 mL) was heated at 60 °C with stirring under nitrogen for 30 min. The reaction mixture was cooled on ice, and a solution of 9 (1.77 g, 4.01 mmol) in THF (5 mL) was added. The mixture was stirred for 1 h, heated at 50 °C for 10 min, cooled, and poured into an aqueous solution of ammonium chloride. Extraction of this mixture with ethyl acetate (3 × 100 mL) and evaporation of the extracts gave the crude bromohydrin (1-bromo3-[7-bromo-1-[(2R)-2-[tert-butyl(dimethyl)silyl]oxypropyl]indazol-6yl]oxy-propan-2-ol) as an oil (1.88 g, 90%). 1H NMR (CDCl3, 200 MHz): δ 7.93 (s, 1H), 7.6 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 5.01 (dd, J = 8, 14 Hz, 1H), 4.54 (dd, J = 6, 14 Hz, 1H), 4.37− 4.18 (m, 4H), 3.72 (m, 2H), 2.75 (d, J = 4.0 Hz, 1H), 1.18 (d, J = 6.0 Hz, 3H), 0.71 (s, 9H), −0.14 (s, 3H), −0.44 (s, 3H); 13C NMR (DMSO-d6, 50 mHz) δ 154.1, 137.9, 133.4, 121.6, 121.0, 109.9, 71.8, 68.4, 68.2, 56.3, 36.7, 25.4, 21.0, 17.3, −5.1, −5.7. This oil was dissolved in dichloromethane (50 mL) and cooled on ice. To the cold solution was added p-toluenesulfonic acid (0.05 g) and ethyl vinyl ether (1 mL, 10.5 mmol) with stirring; after 30 min, TLC showed no starting material. The reaction mixture was poured into a saturated aqueous solution of sodium bicarbonate (100 mL) and extracted with dichloromethane (3 × 50 mL). The combined extracts were dried, filtered, and evaporated to dryness to give a crude oil; column chromatography (silica, gradient, ethyl acetate/hexane 6% to 8%) provided 10 as an oil (1.79 g, 81%): LC-MS (APCI) m/z 593, 595, 597 [M + H]+; 1H NMR (CDCl3, 200 MHz): δ 7.92 (s, 1H), 7.6 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 5.04−4.94 (m, 2H), 4.61− 4.58 (m, 1H), 4.35−4.24 (m, 4H), 3.77−3.58 (m, 4H), 1.43 (m, 2H), 1.28−1.17 (m, 7H), 0.70 (s, 9H), −0.14 (s, 3H), −0.41 (m, 3H). 1-[(2S)-2-Azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol (12). To a stirred solution of 10 (1.78 g, 2.99 mmol) in anhydrous THF (100 mL) under nitrogen at −78 °C was added via syringe a 2.5 M solution of n-butyllithium in hexanes (1.56 mL, 3.90 mmol). The reaction mixture was stirred for 1 h and quenched by the addition of a saturated aqueous solution of sodium bicarbonate (100 mL); this mixture was allowed to warm to room temperature and extracted with ethyl acetate (3 × 100 mL). The combined extracts were dried, filtered, and evaporated to dryness to give a crude oil (1.3 g): LC-MS (APCI) m/z 435 [M + H]+. The oil (0.90 g, 2.07 mmol) was mixed with anhydrous THF (50 mL) and to the solution was added a 1 M solution of tetra-n-butylammonium fluoride in THF (3.11 mL, 3.11 mmol). The mixture was stirred overnight, evaporated to dryness, mixed with a saturated aqueous solution of sodium bicarbonate (50 mL), and extracted with ethyl acetate (3 × 50 mL). The combined extracts were dried, filtered, and evaporated to give a crude oil; column chromatography (silica, gradient, ethyl acetate/hexane 10% to 40%) gave 11 ((2R)-1-[8-(1-ethoxyethoxy)-8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl]propan-2-ol) as an oil (0.35 g, 53%): LC-MS (APCI) m/z 321 [M + H]+; 1H NMR (CDCl3, 200 MHz): δ 7 0.89 (s, 1H), 7.44

investigation of what involvement each of the numerous signaling events initiated by 5-HT2 receptor subtype activation might have in this favorable response is warranted to gain a better understanding of the specific biochemical events that lead to the reduction of IOP in primates for this class of compounds; perhaps leading to the identification of more specific targets to pursue for the treatment of ocular hypertension.

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EXPERIMENTAL METHODS

General. All starting materials and reagents were purchased from commercial suppliers; these were used without further purification following the confirmation of identity. Melting points were determined in open capillaries using a Thomas-Hoover Uni-Melt Apparatus and are uncorrected. Organic extracts were dried with magnesium sulfate. Chromatography refers to column chromatography conducted on 230−400 mesh silica gel from E. Merck. Silica gel TLC plates were obtained from EM Separation Technology. 1H NMR and 13C NMR spectra were determined on either a Bruker AMX 200 MHz spectrometer, Bruker AVANCE III 400 MHz spectrometer, or a Bruker DRX 600 MHz spectrometer. Chemical shift values are reported in parts per million (δ) relative to tetramethylsilane as internal standard. Mass spectra were obtained on a Finnigan TSQ45 triple quadrupole mass spectrometer, and HPLC-MS analyses were recorded on a Finnigan LCQ Classic mass spectrometer. Optical rotations were determined using a Jasco DIP-370 Polarimeter. All compounds subjected to biological testing showed >95% purity as determined by combustion analysis. Elemental analyses were performed by Atlantic Microlabs, Norcross, Georgia and are within ±0.4% of the theoretical values. Evaporations were performed under reduced pressure on a rotary evaporator at 40 °C unless otherwise indicated. 7-Bromo-1-[(2R)-2-[tert-butyl(dimethyl)silyl]oxypropyl]indazol-6ol (8). A solution of (2R)-1-(6-benzyloxyindazol-1-yl)propan-2-ol (5)10 (9.41 g, 0.0334 mol) in THF (50 mL, dry) was prepared under nitrogen at room temperature. Imidazole (4.54 g, 0.0667 mol) and tertbutyldimethylchlorosilane (8.05 g, 0.0534 mol) were added to this solution, and the reaction mixture was stirred at room temperature for 18 h. The reaction mixture was treated with saturated aqueous sodium bicarbonate (300 mL) and extracted with ethyl acetate (2 × 200 mL), and the combined organic layers were dried, filtered, and evaporated to a yellow oil; column chromatography (silica, gradient, ethyl acetate/ hexane; 1:9 to 2:8) gave 6 ([(1R)-2-(6-benzyloxyindazol-1-yl)-1methyl-ethoxy]-tert-butyl-dimethyl-silane) as a yellow oil (12.4 g, 93%). Single compound by LC-MS (APCI) m/z 397 [M + H]+; 1H NMR (CDCl3, 400 MHz) δ 7.79 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.39−7.25 (m, 4H), 7.16 (s, 1H), 6.80−6.75 (m, 2H), 5.02 (s, 2H), 4.23 (m, 1H), 4.12 (m, 2H), 1.12 (d, J = 4.0 Hz, 3H), 0.65 (s, 9H), −0.23 (s, 3H), −0.54 (s, 3H). To a solution of 6 (28.5 g, 0.072 mol) in methanol (280 mL) was added 10% Pd/C (2.8 g), and this mixture was maintained under hydrogen (1 atm, balloon) with stirring for 2 days. A white solid was observed in the reaction mixture; this solid was dissolved by the addition of acetone (250 mL), and the catalyst was removed by filtration through diatomaceous earth. Evaporation of the filtrate gave 7 (1-[(2R)-2-[tert-butyl(dimethyl)silyl]oxypropyl]indazol-6-ol) as a gray solid (20.3 g, 92%): mp 171−172 °C, which was a single compound by LC-MS (APCI) m/z 307 [M + H]+; 1H NMR (DMSOd6, 400 MHz) δ 9.50 (s, 1H), 7.83 (d, J = 4.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 6.76 (s, 1H), 6.62 (m, 1H), 4.14 (m, 3H), 1.14 (s, 3H), 0.67 (s, 9H), −0.19 (s, 3H), −0.48 (s, 3H). To a stirred solution of 7 (3.79 g, 12.4 mmol) in anhydrous THF (100 mL) at 0 °C was added N-bromosuccinimide (2.20 g, 12.4 mmol). The reaction mixture was stirred for 30 min, quenched by the addition of a saturated aqueous solution of sodium thiosulfate (50 mL), and extracted with ethyl acetate (3 × 100 mL). The combined extracts were washed with a saturated aqueous solution of sodium bicarbonate (100 mL) and dried. Column chromatography (silica, gradient, ethyl acetate/hexane) gave 8 as a solid (3.66 g, 77%): mp I

DOI: 10.1021/acs.jmedchem.5b00857 J. Med. Chem. XXXX, XXX, XXX−XXX

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filtrate evaporated to a dark yellow oil (12.2 g); column chromatography (silica gel, gradient, ethyl acetate/hexane, 8:2 to 7:3) gave 15 (tert-butyl-dimethyl-[(1R)-1-methyl-2-(6-prop-2-ynoxyindazol-1-yl)ethoxy]silane) as a yellow oil (10.9 g, 89%): LC-MS (APCI) m/z 345 [M + H]+. 1H NMR (CDCl3, 600 MHz): δ 7.89 (s, 1H), 7.56 (d, J = 6.0 Hz, 1H), 6.89 (s, 1H), 6.83 (dd, J = 6, 12 Hz, 1H), 4.75 (s, 2H), 4.33 (m, 1H), 4.22 (m, 2H), 2.54 (m, 1H), 1.22 (d, J = 6 Hz, 3H), 0.74 (s, 9H), −0.12 (s, 3H), −0.41 (s, 3H). A suspension of 15 (10.94 g, 0.0318 mol) in mesitylene (60 mL) was degassed with reduced pressure and nitrogen, followed by heating the mixture at 200 °C in a sealed glass pressure vessel for 2 h and 190 °C for 18 h. Column chromatography of the brown solution (silica gel, hexane/ethyl acetate 9:1) gave 16 as a yellow solid (9.53 g, 87%): mp 58−59 °C; LC-MS (APCI) m/z 345 [M + H]+. 1H NMR (CDCl3, 600 MHz): δ 7.86 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 11 Hz, 1H), 6.70 (d, J = 9.0 Hz, 1H), 5.83 (dt, J = 4.2, 9.6 Hz, 1H), 4.73 (m, J = 4.2, 13.8, 24.0 Hz, 2H), 4.45−4.30 (m, 2H), 4.36 (m, 1H), 1.26 (d, J = 6.0 Hz, 3H), 0.71 (s, 9H), −0.16 (s, 3H), −0.46 (s, 3H). (8R)-1-[(2R)-2-[tert-Butyl(dimethyl)silyl]oxypropyl]-8,9-dihydro7H-pyrano[2,3-g]indazol-8-ol (17a). To a solution of 16 (1.0 g, 2.91 mmol) in anhydrous THF (80 mL) was added a 0.5 M solution of 9BBN in THF (1.3 mL, 6.4 mmol) and heated at reflux temperature for 2 h. The mixture was cooled on ice, and the reaction quenched by the addition of MeOH (5 mL) and then 30% H2O2 (5 mL) over about 1 min. The mixture was stirred for 1 h at ambient temperature until one major polar product was detected on TLC. The volatiles were evaporated, and the residue was mixed with a saturated solution of sodium bicarbonate (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined extracts were dried, filtered, and concentrated to give a crude oil (NMR showed a 9:1 mixture of diastereomers). Column chromatography (silica, gradient 10% to 30% ethyl acetate/ hexane) gave 17a [R-C8-OH] as an oil (0.56g, 53%): LC-MS (APCI) m/z 363 [M + H]+. 1H NMR (DMSO-d6, 600 MHz): δ 7.90 (s, 1H), 7.40 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 8.0 Hz, 1H), 5.25 (s, 1H), 4.45− 4.29 (m, 2H), 4.21 (m, 1H), 4.05 (m, 2H), 3.77 (m, 1H), 3.58 (m, 1H), 2.82 (m, 1H), 1.21 (d, J = 6.0 Hz, 3H), 0.61 (s, 9H), −0.22 (s, 3H), −0.64 (s, 3H). Also obtained was an oil (0.22 g) that consisted of a mixture of the two unresolved isomers: 17a, R-C8-OH and 17b: SC8-OH. (8R)-1-[(2S)-2-Azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol (12a). To a mixture of 17a (0.56 g, 1.55 mmol) and pyridinium p-toluenesulfonate (0.05 g) in methylene chloride (50 mL) at 0 °C was added ethyl vinyl ether (1 mL). After 1 h, the ice bath was removed, and the mixture was stirred for additional 1 h. TLC showed a less polar compound. TEA (1 mL) was added to the mixture and evaporated to dryness, and anhydrous THF (10 mL) was added, followed by tetra-n-butylammonium fluoride (1 M, 3.1 mL). The mixture was stirred for 1 h and evaporated to give a crude product. Column chromatography (silica, gradient, 10% to 30% ethyl acetate/ hexane) gave 18 ((2R)-1-[(8R)-8-(1-ethoxyethoxy)-8,9-dihydro-7Hpyrano[2,3-g]indazol-1-yl]propan-2-ol) as an oil (0.47 g, 96%): LCMS (APCI) m/z 321 [M + H]+. 1H NMR (CDCl3, 600 MHz): δ 7.88 (s, 1H), 7.43 (d, J = 6.0 Hz, 1H), 6.73 (d, J = 6.0 Hz, 1H), 4.94 (d, J = 6.0 Hz, 1H), 4.52−4.39 (m, 2H), 4.32−4.25 (m, 3H), 3.99 (m, 1H), 3.68 (m, 1H), 3.59 (m, 3H), 3.05 (m, 1H), 1.39 (d, J = 5.4 Hz, 3H), 1.29 (m, 3H), 1.24 (t, J = 7.2 Hz, 3H). To a solution of 18 (0.46 g, 1.44 mmol) in anhydrous THF (50 mL) at 0 °C was added triethylamine (0.73 g, 1.0 mL, 7.19 mmol) and methanesulfonic anhydride (0.50 g, 2.88 mol) with stirring. The ice bath was removed, and the reaction was stirred for about 20 min. The mixture was concentrated to dryness, and the residue was mixed with sodium azide (1.05 g, 16.1 mmol) and anhydrous DMF (50 mL). The mixture was heated at 100 °C for about 5 h, cooled, and mixed with a saturated aqueous sodium bicarbonate (100 mL) and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were dried, filtered, and evaporated to a yellow residue. Column chromatography (gradient, 1% to 10% ethyl acetate/hexane) gave an oil (0.38 g, 77%), which was dissolved in MeOH (10 mL), and treated with TsOH (cat.) for 1 h at ambient temperature followed by the addition of TEA (0.1 mL); this mixture was stirred for 5 min and evaporated to a residue.

(d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 4.97 (m, 1H), 4.52−3.98 (m, 6H), 3.69−3.03 (m, 5H), 1.41−1.18 (m, 9H). To a stirred solution of 11 (0.82 g, 2.56 mmol) in anhydrous THF (80 mL) at 0 °C was added triethylamine (1.29 g, 12.8 mmol) and methanesulfonic anhydride (1.11 g, 6.40 mmol); this mixture was stirred for 1 h and evaporated to dryness. The residue was mixed with anhydrous DMF (100 mL) and sodium azide (3.32 g, 51.2 mmol) followed by heating at 95 °C for 7 h. The reaction mixture was cooled and mixed with a saturated aqueous solution of sodium bicarbonate (200 mL), extracted with ethyl acetate (3 × 100 mL), and the combined extracts were evaporated to give the azide as an oil (0.72 g, 81%). A solution of this oil in THF (50 mL) containing 2 N HCl (10 mL) was stirred at room temperature for 30 min and then poured into a saturated aqueous solution of sodium bicarbonate (100 mL); this mixture was extracted with ethyl acetate (3 × 50 mL). The combined extracts were dried, filtered, and evaporated to give a crude oil; column chromatography (silica, gradient, ethyl acetate/hexane 20% to 40%) gave 12 as an oil (0.85 g, 79%): LC-MS (APCI) m/z 274 [M + H]+. 1 H NMR (DMSO-d6, 600 MHz): δ7.95 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 5.22 (t, J = 0.72 Hz, 1H), 4.54−4.45 (m, 2H), 4.11 (m, 3H), 3.86 (m, 1H), 3.36 (m, 1H), 2.97 (m, 1H), 1.24 (d, J = 6.6 Hz, 3H). (8R)-1-[(2S)-2-Azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol (12a) and (8S)-1-[(2S)-2-Azidopropyl]-8,9-dihydro-7Hpyrano[2,3-g]indazol-8-ol (12b). The diastereomeric mixture 12 (1.35 g) was separated (Chiral Technologies, Inc.) using a CHIRACEL OJ column: eluent 2-propanol/hexane 1:9 at 25 °C. UV detector, 235 nm: 12b (0.679 g) and 12a (0.653 g), the first (6.09 min) and second (6.97 min) peaks, respectively. For the final analysis of diastereomeric purity, a CHIRACEL OD, 10 μm column was used with ethanol/hexane (5/95) as mobile phase: 12b and 12a showed de of 99.5% and 97.8%, respectively. (Note: Under the final analytical conditions, the order of elution of the two diastereomers was reversed relative to that observed for the prep column.) (8R)-1-[(2S)-2-Aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol (13). Diastereomer 12a (0.23 g) was dissolved in MeOH and Pd/C (10%, 0.04 g) added; this mixture was placed under hydrogen at atmospheric pressure for 16 h. The catalyst was removed by filtration, washed with MeOH, and the filtrate evaporated to give 13 (0.21 g). Crystallization from methanol/ethyl acetate gave a pale yellow solid: mp 126−128 °C; [α]D = +47.7 (0.352%, MeOH); [α]405 = +112 (0.352%, MeOH); LC-MS (APCI) m/z 248 [M + H]+. 1H NMR (CDCl3, 600 MHz): δ 7.85 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 4.39 (d, J = 4.2 Hz, 1H), 4.29 (m, 2H), 4.11 (m, 2H), 3.45 (m, 1H), 3.28 (dd, J = 4.8, 11.4 Hz, 2H), 1.12 (d, J = 6.6 Hz, 3H). 13C NMR (CDCl3, 100 mHz): δ 153.32, 139.97, 133.93, 119.94, 119.93 (C4), 113.63, 100.56, 69.22, 62.53, 58.75, 48.45, 30.89, 20.90. Anal. (C13H17N3O2) C, H, N. Crystals suitable for X-ray analysis were obtained from 2-propanol; this analysis established the absolute stereochemistry of R for the hydroxyl group at position C8 (see Supporting Information.) (8S)-1-[(2S)-2-aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-ol (14). Treatment of diasteroisomer 12b using the same procedure as that described above for the preparation of 13 provided amine 14 as an oil: LC-MS (APCI) m/z 248 [M + H]+. 1H NMR (DMSO-d6, 200 MHz): δ 7.89 (s, 1H), 7.42 (d, J = 8.6 Hz, 1H,), 6.62 (d, J = 8.6 Hz, 1H), 4.26 (m, 2H), 4.09 (m, 2H), 3.84 (m, 1H), 3.45− 2.85 (m, 6H), 0.94 (d, J = 6.4 Hz, 3H); [α]D = −6.21 (0.467%, MeOH); [α]405 = −13.5 (0.467%, MeOH). Anal. (C13H17N3O2· 0.2 H2O) C, H, N. Alternate Synthesis of (8R)-1-[(2S)-2-Azidopropyl]-8,9-dihydro7H-pyrano[2,3-g]indazol-8-ol (12a). tert-Butyl-dimethyl-[(1R)-1methyl-2-(7H-pyrano[2,3-g]indazol-1-yl)ethoxy]silane (16). A solution of 7 (10.9 g, 0.0357 mol) in acetone (250 mL) was degassed with reduced pressure and nitrogen; pulverized potassium carbonate (6.90 g, 0.0357 mol) and propargyl bromide (5.19 mL, 0.0464 mol) were added to the solution, and the mixture was refluxed for 18 h. Additional potassium carbonate (1.97 g, 0.014 mol) and propargyl bromide (1.2 mL, 0.0107 mol) were added, and the mixture was refluxed an additional 2 h. The reaction mixture was filtered, and the J

DOI: 10.1021/acs.jmedchem.5b00857 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Column chromatography (gradient, 10% to 35% ethyl acetate/hexane) gave 12a as a solid (0.27 g, 99%): LC-MS (APCI) m/z 274 [M + H]+; 1 H NMR (DMSO-d6, 600 MHz): δ 7.96 (s, 1H), 7.45 (d, J = 9.0 Hz, 1H), 6.64 (d, J = 9.0 Hz, 1H), 5.22 (s, 1H), 4.54−4.43 (ddd, J = 4.2, 8.4, 14.4 Hz, 2H), 4.12 (m, 3H), 3.86 (dd, J = 6.6, 10.2 Hz, 1H), 3.41 (dd, J = 5.4, 15.6 Hz, 1H), 2.92 (dd, J = 6.0, 15.6 Hz, 1H), 1.25 (d, J = 6.6 Hz, 3H). (8R)-8-Azido-1-[(2S)-2-azidopropyl]-8,9-dihydro-7H-pyrano[2,3g]indazole (19). To a stirred solution of 12b (0.44 g, 1.61 mmol) in anhydrous THF (50 mL) at 0 °C was added triethylamine (0.65 g, 6.44 mmol) and methanesulfonic anhydride (0.56 g, 3.22 mmol). The mixture was stirred for 1 h and evaporated to dryness. The residue was mixed with anhydrous DMSO (50 mL), sodium azide (1.05 g, 16.1 mmol), and heated at 90 °C for 5 h. The reaction mixture was cooled and mixed with a saturated aqueous solution of sodium bicarbonate (100 mL), extracted with ethyl acetate (3 × 80 mL), and evaporated to give an oil. Column chromatography (silica, gradient of ethyl acetate/ hexane (5% to 25%)) gave the azide 19 as an oil (0.19 g, 40%): LCMS (ESI) m/z 299 [M + H]+. 1H NMR (CDCl3, 600 MHz): δ7.92 (s, 1H), 7.46 (d, J = 12 Hz, 1H), 6.75 (d, J = 12 Hz, 1H), 4.44 (m, 2H), 4.27 (d, J = 12 Hz, 1H), 4.16−4.09 (m, 3H), 3.38 (m, 2H), 1.35 (d, J = 6.0 Hz, 3H). (8R)-1-[(2S)-2-Aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-amine (20). The oil 19 (0.19 g, 0.64 mmol) was dissolved in MeOH, and Pd/C (10%, 0.019 g) was added; this mixture was placed under hydrogen (1 atm) for 16 h. The reaction mixture was filtered and the filtrate evaporated to give an oil, which was dissolved in MeOH and treated with 2 N HCl/EtOH. Evaporation gave a crude solid that was triturated with a mixture of methanol in dichloromethane to give 20 as a pale yellow solid (0.16 g, 98%): mp >300 °C; LC-MS (ESI) m/z 247 [M + H]+. 1H NMR (DMSO-d6, 600 MHz): δ 8.66 (br s, 3H), 8.45 (br s, 3H), 8.04 (s, 1H), 7.56 (d, J = 6.0 Hz, 1H), 6.75 (d, J = 6.0 Hz, 1H), 4.75 (m, 1H), 4.61 (m, 1H), 4.31 (m, 2H), 3.86 (br s, 1H), 3.79 (br t, 1H), 3.64 (m, 1H), 3.46 (m, 1H), 1.15 (d, J = 6.0 Hz, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 152.7, 139.2, 134.3, 120.0, 119.6, 113.6, 99.9, 65.1, 52.5, 47.2, 42.6, 24.9, 15.9. Anal. (C13H18N4O·3HCl·0.33 C2H5OH·0.5 H2O) C, H, N. (2R)-1-[(8R)-8-(2-Hydroxyethoxy)-8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl]propan-2-ol (23). To a stirred solution of 17a (2.05 g, 5.66 mmol) in anhydrous DMF (60 mL) at 0 °C was added sodium hydride (60% dispersion in mineral oil, 0.34 g, 8.49 mmol). The mixture was stirred for 20 min, and to this solution was added t-butyl bromoacetate (1.32 g, 6.79 mmol) and sodium iodide (0.1 g). The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was poured into a saturated aqueous solution of sodium bicarbonate (100 mL) and extracted with ethyl acetate (3 × 100 mL). The combined extracts were dried, filtered, and evaporated to dryness. Column chromatography (silica, gradient of ethyl acetate/ hexane (5% to 25%) gave 21 (tert-butyl 2-[[(8R)-1-[(2R)-2-[tertbutyl(dimethyl)silyl]oxypropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol8-yl]oxy]acetate) as an oil (2.11 g, 78%): LC-MS (APSI) m/z 477 [M + H]+; 1H NMR (CDCl3, 400 MHz) δ 7.85 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H), 4.53−4.25 (m, 4H), 4.15 (s, 2H), 4.03 (m, 2H), 3.80 (m, 1H), 3.02 (m, 1H), 1.50 (s, 9H), 1.25 (d, J = 8.0 Hz, 3H), 0.66 (s, 9H), −0.20 (s, 3H), −0.62 (s, 3H). To a stirred solution of 21 (2.60 g, 4.41 mmol) in anhydrous THF (50 mL) was added a solution of tetra-n-butylammonium fluoride (1 M in THF, 5.29 mL, 5.29 mmol). The mixture was stirred overnight at room temperature, evaporated to dryness, mixed with a saturated aqueous solution of sodium bicarbonate (50 mL), and extracted with ethyl acetate (3 × 60 mL). The combined extracts were dried, filtered, and evaporated to give 22 (tert-butyl 2-[[(8R)-1-[(2R)-2-hydroxypropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]acetate) as an oil (1.32 g, 82%): LC-MS (APCI) m/z 363 [M + H]+; 1H NMR (CDCl3, 400 MHz) δ 7.88 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 4.52 (m, 1H), 4.41−4.25 (m, 3H), 4.16 (d, J = 2.4 Hz, 2H), 4.15−4.04 (m, 2H), 3.56 (m, 1H), 3.50 (d, J = 3.2 Hz, 1H), 3.18 (m, 1H), 1.49 (s, 9H), 1.28 (d, J = 6.0 Hz, 3H). To a stirred solution of 22 (0.65 g, 1.80 mmol) in anhydrous THF at 0 °C was added lithium aluminum hydride (1 M in THF, 1.80 mL,

1.80 mmol). The mixture was stirred at room temperature for 1 h, cooled on ice, and quenched by the addition of 2 N HCl. The mixture was extracted with ethyl acetate (3 × 60 mL), dried, filtered, and evaporated to dryness. Column chromatography (silica, gradient, 5% to 10% ethyl acetate in hexane) gave 23 ((2R)-1-[(8R)-8-(2hydroxyethoxy)-8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl]propan-2ol) as an oil (0.37 g, 71%): LC-MS (APCI) m/z 293 [M + H]+; 1H NMR (CDCl3, 400 MHz) δ 7.88 (s, 1H), 7.43 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 4.53 (dd, J = 4.0, 12 Hz, 2H), 4.36 (m, 1H), 4.12 (m, 2H), 4.01 (m, 1H), 3.49 (m, 1H), 3.37 (d, J = 3.6 Hz, 1H), 3.13 (dd, J = 5.6, 16 Hz, 1H), 1.99 (br s, 1H), 1.28 (d, J = 6.0 Hz, 3H). (2S)-1-[(8R)-8-(2-Aminoethoxy)-8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl]propan-2-amine (25). To a stirred solution of 23 (0.37 g, 1.27 mmol) in anhydrous THF (20 mL) at 0 °C was added triethylamine (0.64 g, 6.34 mmol) and methanesulfonic anhydride (0.66 g, 3.81 mmol). The mixture was stirred for 30 min, evaporated to dryness, and mixed with anhydrous dimethyl sulfoxide (30 mL) and sodium azide (0.83 g, 12.7 mmol). The mixture was heated at 90 °C for 4 h, cooled, mixed with a saturated aqueous solution of sodium bicarbonate (50 mL), and extracted with ethyl acetate (3 × 50 mL). The combined extracts were filtered and evaporated to give 24 ((8R)8-(2-azidoethoxy)-1-[(2S)-2-azidopropyl]-8,9-dihydro-7H-pyrano[2,3g]indazole) as an oil (0.36 g, 84%): LC-MS (ESI) m/z 343 [M + H]+; 1 H NMR (CDCl3, 400 MHz) δ 7.91 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 4.46 (m, 2H), 4.22 (m, 1H), 4.12 (m, 3H), 3.82 (m, 2H), 3.42 (m, 4H), 1.32 (d, J = 6.4 Hz, 3H). To a solution of 24 (0.36 g, 1.05 mmol) in methanol (10 mL) was added Pd/C (10%, 0.03 g) and the mixture stirred under hydrogen (1 atm) for 2 days followed by filtration and evaporation of the filtrate to an oil. The oil was dissolved in methanol (5 mL), and 2 N HCl/EtOH (3 mL) was added followed by evaporation to a solid that was triturated with methanol/dichloromethane (1:10), filtered, and dried to give 25 as an off-white solid (0.25 g, 60%): mp 282−285 °C; LCMS (APCI) m/z 291 [M + H]+; 1H NMR (DMSO-d6, 400 MHz) δ 8.46 (br s, 3H), 8.14 (br s, 2H), 8.01 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 4.80 (dd, J = 4.0, 12 Hz, 1H), 4.64 (dd, J = 8.0, 12 Hz, 1H), 4.17 (s, 2H), 4.05 (t, J = 4.0 Hz, 1H), 3.89−3.82 (m, 3H), 3.50 (dd, J = 4.0, 16 Hz, 1H), 3.27 (dd, J = 4.4, 17 Hz, 1H), 3.00 (br s, 2H), 1.15 (s, 3H); 13C NMR (DMSO-d6, 100 mHz) δ 153.1, 139.7, 134.3, 119.4, 119.2, 113.3, 101.2, 69.24, 65.7, 64.4, 52.4, 47.3, 38.7, 27.1, 15.9. Anal. (C15H22N4O2·2 HCl·0.25 H2O) C, H, N. tert-Butyl 2-[[(8R)-1-[(2S)-2-azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]acetate (26). A solution of 22 (0.4 g, 1.33 mmol) in THF (14 mL) was cooled in an ice bath, triethylamine (0.74 mL, 5.3 mmol) and methanesulfonic anhydride (0.42 g, 2.39 mmol) were added, and the mixture was stirred for 40 min followed by the addition of sodium azide (0.86 g, 13.3 mmol) and evaporation to a residue, which was taken up in DMSO (14 mL) and stirred at 90 °C for 2.5 h. The reaction mixture was cooled to room temperature and quenched with brine (50 mL); this mixture was extracted with ethyl acetate (3 × 100 mL). The combined extracts were dried, filtered, and evaporated to give a colorless oil (0.49 g); column chromatography (silica gel, hexane/ethyl acetate) gave 26 as a colorless solid: (0.42 g, 82%); mp 72−74 °C; LC-MS (APCI) m/z 388 [M + H]+; 1H NMR (CDCl3, 400 MHz) δ7.90 (s, 1H), 7.45 (d, J = 6 Hz, 1H), 6.70 (d, J = 6 Hz, 1H), 4.5 (m, 2H), 4.3 (m, 1H), 4.1 (m, 5H), 3.35 (m, 2H), 1.50 (s, 9H), 1.32 (d, J = 4 Hz, 3H). 2-[[(8R)-1-[(2S)-2-Aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]ethanol (28). A solution of 26 (1.2 g, 3.1 mmol) in THF (30 mL) was cooled in an ice bath, and a 1 N solution of lithium aluminum hydride in THF (6.2 mL, 6.2 mmol) was added, and the reaction mixture was allowed to warm to room temperature with stirring. After stirring for 1 h, the reaction mixture was cooled in an ice bath and quenched by the dropwise addition of 2 N aqueous potassium hydroxide (0.317 mL). The solids were filtered off, and the filtrate was evaporated to give 28 as a pale yellow oil (0.76 g, 84%), which was about 90% pure by LC-MS (APCI) m/z 292 [M + H]+. To facilitate purification, a solution of this crude material (0.76 g, 2.61 mmol) in ethyl acetate (50 mL) and benzyl chloroformate (0.39 mL, 2.61 mmol) was treated with aqueous saturated sodium bicarbonate K

DOI: 10.1021/acs.jmedchem.5b00857 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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temperature for 1 day. The reaction mixture was filtered and evaporated to give 32b as a yellow syrup (0.12 g (86%): LC-MS (APCI) m/z 363 [M + H]+; 1H NMR (CD3OD, 400 MHz) δ 7.8 (s, 1H), 7.3 (d, J = 8.8 Hz, 1H), 6.6 (d, J = 8.8 Hz, 1H), 4.3 (d, J = 6 Hz, 2H), 4.18 (m, 1H), 4.07 (m, 3H), 4.04 (m, 1H), 3.3 (m, 7H), 3.2 (m, 3H), 0.98 (d, J = 6 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 172.6, 155.2, 141.3, 135, 121.3, 121, 115, 102.1, 72.3, 71.8, 69.1, 67.1, 59.1, 58.9, 39.7, 28.9, 20.5. Anal. (C18H26N4O4·0.5 H2O) C, H, N. N-(3-Aminopropyl)-2-[[(8R)-1-[(2S)-2-aminopropyl]-8,9-dihydro7H-pyrano[2,3-g]indazol-8-yl]oxy]acetamide (32c). A solution of 30 (0.17 g, 0.49 mmol) in methanol (10 mL) was stirred with 1,3propanediamine (2 mL, 24 mmol) at room temperature for 18 h. The reaction was evaporated to a yellow residue; column chromatography (silica gel, 10% methanol/dichloromethane/1% triethylamine) gave 31c (N-(3-aminopropyl)-2-[[(8R)-1-[(2S)-2-azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]acetamide) as a yellow oil (0.17 g, 89%): LC-MS (APCI) m/z 388 [M + H]+. A solution of 31c (170 mg, 0.44 mmol) in methanol (10 mL) was stirred with Pd/C (10%, 20 mg) under hydrogen (1 atm) at room temperature for 1 day. The reaction mixture was filtered and evaporated to give 32c as a colorless oil (0.15 g, 95%): LC-MS (APCI) m/z 362 [M + H]+; 1H NMR (CD3OD, 400 MHz) δ 7.8 (s, 1H), 7.42 (d, J = 8.8 Hz, 1H), 6.6 (d, J = 8.8 Hz, 1H), 4.33 (d, J = 6 Hz, 2H), 4.2 (m, 1H), 4.05 (m, 3H), 4.02 (m, 1H), 3.25 (m, 7H), 2.45 (t, J = 7 Hz, 1H), 1.75 (m, 1H), 1.45 (m, 2H), 1.24 (m, 1H), 0.98 (d, J = 6 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 172.6, 155.2, 141.2, 134.9, 121.2, 120.9, 114.9, 102.1, 72.2, 68.9, 67.1, 66.9, 59.1, 40.8, 39.7, 37.3, 33.4, 28.8, 20.5, 7.6. Anal. (C18H27N5O3·0.5 CH3OH) C, H, N. 2-[[(8R)-1-[(2S)-2-aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]-1-[4-(2-pyridyl)piperazin-1-yl]ethanone (32d). To 30 (0.21 g, 0.61 mmol) was added 1-(pyridin-2-yl)piperazine (1 mL, 6.9 mmol), and this mixture was stirred at 60 °C for 18 h followed by heating at 90 °C for 2 h, and finally stirring at 60 °C for 3 days. The reaction mixture was cooled to room temperature, and water (100 mL) was added. The mixture was extracted into ethyl acetate (3 × 100 mL), and the combined organic phases were washed with water (2 × 100 mL), dried, filtered, and evaporated to a yellow oil; column chromatography (silica, ethyl acetate) gave 31d (2-[[(8R)-1-[(2S)-2azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]-1-[4-(2pyridyl)piperazin-1-yl]ethanone) as a colorless oil (0.19 g, 66%): LCMS (APCI) m/z 477 [M + H]+. A solution of 31d (190 mg, 0.40 mmol) in methanol (10 mL) was stirred with Pd/C (10%, 20 mg) under hydrogen (1 atm) at room temperature for 24 h. The reaction mixture was filtered and evaporated to give 32d as a white solid (0.18 g, 98%): LC-MS (APCI) m/z 451 [M + H]+; 1H NMR (CD3OD, 400 MHz) δ 7.98 (m, 1H), 7.75 (s, 1H), 7.46 (m, 1H), 7.29 (d, J = 8.4 Hz, 1H), 6.6 (m, 3H), 4.41 (m, 1H), 4.25 (m, 3H), 4.05 (m, 2H), 3.45 (m, 6H), 3.25 (m, 4H), 3.1 (m, 2H), 0.97 (d, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 170.5, 160.7, 155.2, 148.5, 141.3, 139.2, 134.9, 121.2, 120.9, 115, 114.9, 109.2, 101.9, 72, 69.2, 67.2, 59.2, 46.6, 46.1, 45.9, 42.7, 28.7, 20.4. Anal. (C24H30N6O3·0.58 H2O) C, H, N. X-ray Crystallographic Analysis of 13. Crystals of 13 grew as clusters of colorless needles from 2-propanol. The data crystal was cut from a larger needle and had approximate dimensions of 0.39 × 0.29 × 0.22 mm3. The data were collected on a Nonius Kappa CCD diffractometer using a graphite monochromator with MoKα radiation (λ = 0.71073 Å). A total of 330 frames of data were collected using ωscans with a scan range of 1° and a counting time of 32 s per frame. The data were collected at 153 K using an Oxford Cryostream low temperature device. Details of crystal data, data collection, and structure refinement are listed in (Table S3). Data reduction was performed using DENZO-SMN.30 The structure was solved by direct methods using SIR9231 and refined by full-matrix least-squares on F2 with anisotropic displacement parameters for the non-H atoms using SHELXL-97.32 The hydrogen atoms were observed in a ΔF map and refined with isotropic displacement parameters. The function, Σw(|Fo|2 − |Fc|2)2, was minimized, where w = 1/[(σ(Fo))2 + (0.0206·P)2 + (0.094·P)], and P = (|Fo|2 + 2|Fc|2)/3. Rw(F2) refined to 0.0642, with R(F) equal to 0.0260 and a goodness of fit, S, = 1.065. Definitions used for calculating R(F),Rw(F2) and the goodness of fit, S, are provided:

(50 mL) with stirring at room temperature for 1 h. The organic layer was collected, and the aqueous layer was extracted with ethyl acetate (2 × 100 mL). The combined organic layers were dried, filtered, and evaporated to give the carbamate 27 (benzyl N-[(1S)-2-[(8R)-8-(2hydroxyethoxy)-8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl]-1-methyl− ethyl]carbamate) as a colorless oil (0.41 g, 37%): LC-MS (APCI) m/z 426 [M + H]+; (CDCl3, 400 MHz) δ 7.83 (s, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.33 (m, 5H), 6.73 (d, J = 8.8 Hz, 1H), 5.25 (br s, 1H), 5.05 (s, 2H), 4.70 (d, J = 10 Hz, 1H), 4.41 (dd, J = 6.8, 14.6 Hz), 4.22−4.02 (m, 4H), 3.81−3.76 (m, 4H), 3.4 (br s, 2H), 1.13 (d, J = 6.4 Hz, 3H). A solution of 27 (0.36 g, 0.85 mmol) in methanol (20 mL) was stirred with 10% Pd/C (36 mg) under hydrogen (1 atm) for 2 days. The reaction mixture was filtered and evaporated to give 28 as a pale yellow syrup: (0.23 g, 93%); LC-MS (APCI) m/z 292 [M + H]+; 1H NMR (CDCl3, 400 MHz) δ 7.84 (s, 1H), 7.39 (d, J = 8.8 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 4.39 (m, 2H), 4.17 (m, 2H), 4.01 (m, 1H), 3.78−3.71 (m, 4H), 3.50 (m, 1H), 3.35 (dd, J = 5.6, 13 Hz, 2H), 1.16 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 155.4, 141.4, 135.1, 121.1, 120.8, 114.9, 102.3, 71.5, 67.6, 62.5, 58.4, 29.3, 19.9. Anal. (C15H21N3O3·0.25 H2O·0.125 CDCl3) C, H, N. Methyl 2-[[(8R)-1-[(2S)-2-azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]acetate (30). A solution of 12a (1.92 g, 7 mmol) in DMF (20 mL) was cooled by an ice bath (0 °C) and was treated with sodium hydride (60% in oil, 0.56 g, 14 mmol). After bubbling ceased, the reaction was treated with isopropyl bromoacetate (1.82 mL, 14 mmol) and allowed to warm to room temperature over 2 h. The reaction mixture was poured into aqueous saturated sodium bicarbonate (150 mL), extracted with ethyl acetate (3 × 100 mL), and the combined organic phases were washed with brine (100 mL) and dried. The filtrate was evaporated to a yellow residue; column chromatography (silica, 20% ethyl acetate/hexane) gave 29 (isopropyl 2-[[(8R)-1-[(2S)-2-azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]acetate) as a colorless oil (1.69 g, 64%): LC-MS (APCI) m/z 374 [M + H]+. A solution of 29 (1.69 g, 4.5 mmol) in methanol (20 mL) was combined with a 3 N solution of hydrochloric acid in methanol (5 mL, 15 mmol) and the mixture refluxed for 18 h. The reaction mixture was evaporated to a colorless residue; column chromatography (silica, 20% ethyl acetate/hexane) gave 30 as a colorless oil (1.13 g, 73%): LC-MS (APCI) m/z 346 [M + H]+. This material was used in subsequent reactions without further purification. 2-[[(8R)-1-[(2S)-2-Aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]-N-methyl-acetamide (32a). A solution of 30 (0.15 g, 0.43 mmol) was treated with a 2 N solution of methylamine in THF (10 mL, 20 mmol) with stirring at room temperature for 18 h. The reaction mixture was evaporated to a yellow oil; column chromatography (silica, gradient, 20% ethyl acetate/hexane to ethyl acetate) gave 31a (2-[[(8R)-1-[(2S)-2-azidopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]-N-methyl-acetamide) as a colorless solid (0.14 g, 94%): LC-MS (APCI) m/z 345 [M + H]+. A solution of 31a (140 mg, 0.4 mmol) in methanol (10 mL) was stirred with Pd/C (10%, 20 mg) under hydrogen (1 atm) at room temperature for 1 day. The reaction mixture was filtered and evaporated to give 32a as a colorless solid (0.13 g, 98%): LC-MS (APCI) m/z 319 [M + H]+; 1H NMR (CD3OD, 400 MHz) δ 7.78 (m, 1H), 7.34 (d, J = 8.8 Hz, 1H), 6.59 (d, J = 8.8 Hz, 1H), 4.34 (d, J = 6 Hz, 2H), 4.13 (m, 1H), 4.05 (m, 3H), 4.0 (m, 1H), 3.28 (m, 3H), 2.62 (s, 3H), 0.97 (d, J = 6 Hz, 3H); 13 C NMR (100 MHz, CD3OD) δ 172.9, 155.2, 141.2, 134.9, 128.4, 121.2, 120.9, 114.9, 102.1, 72.2, 68.9, 67.1, 59.1, 28.9, 25.9, 20.5. Anal. (C16H22N4O3·0.2 CH3OH·0.8 H2O) C, H, N. 2-[[(8R)-1-[(2S)-2-Aminopropyl]-8,9-dihydro-7H-pyrano[2,3-g]indazol-8-yl]oxy]-N-(2-methoxyethyl)acetamide (32b). A solution of 30 (0.15 g, 0.43 mmol) in methanol (10 mL) was stirred with 2methoxyethylamine (2 mL, 23 mmol) at room temperature for 18 h. The reaction mixture was evaporated to a yellow oil; column chromatography (silica gel, gradient, hexane/ethyl acetate, 4:1 to ethyl acetate) gave 31b (2-[[(8R)-1-[(2S)-2-azidopropyl]-8,9-dihydro7H-pyrano[2,3-g]indazol-8-yl]oxy]-N-(2-methoxyethyl)acetamide) as a yellow oil (0.15 g, 90%): LC-MS (APCI) m/z 389 [M + H]+. A solution of 31b (150 mg, 0.39 mmol) in methanol (10 mL) was stirred with Pd/C (10%, 20 mg) under hydrogen (1 atm) at room L

DOI: 10.1021/acs.jmedchem.5b00857 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Rw(F2) = {Σw(|Fo|2 − |Fc|2)2/Σw(|Fo|)4}1/2, where w is the weight given each reflection; R(F) = Σ(|Fo| − |Fc|)/Σ|Fo|} for reflections with Fo > 4(Σ(Fo)); S = [Σw(|Fo|2 − |Fc|2)2/(n − p)]1/2, where n is the number of reflections, and p is the number of refined parameters. The data were checked for secondary extinction effects, but no correction was necessary. The absolute configuration was determined by internal comparison to the known configuration at C15 (see Figure S1 for the atom labeling scheme). Neutral atom scattering factors and values used to calculate the linear absorption coefficient are from the International Tables for X-ray Crystallography.33 All figures were generated using SHELXTL/PC.34 Summary of Crystal Structure Data for 13. Empirical formula, C13H17N3O2; formula weight, 247.30; collection temperature, 153 K; crystal system, monoclinic; space group, P21; unit cell dimensions, a = 9.0838(2) Å, α = 90°; b = 4.4965(1) Å, β = 101.147(1)°; c = 14.9860(3) Å, γ = 90°; Z, 2. Goodness-of-fit on F2, 1.065; final R indices [I > 2σ(I)], R1 = 0.0260, wR2 = 0.0632. Crystallographic data for structure 13 is available free of charge from Cambridge Crystallographic Data Center under deposition number CCDC 1410547. Determination of Compound Stability. The aqueous stability of compounds was conducted in pH 7.4, 0.025 M sodium phosphate buffer. Each compound was dissolved [5 μg/mL (0.0005%) and/or 1%] in buffer, and the solutions were heated at either 75 °C for up to 4 weeks or at 45 °C for up to 12 weeks. Water for injection was used for buffer preparation. For pH adjustment, 0.6 N HCl and 1.0 N NaOH stored in glass containers were used. An HPLC method was developed for the analysis of stability samples of each compound. The stability results (percent degradation) were used for calculation of the predicted half-life of a compound at 25 °C. This prediction was based on the time required for a 10% loss of compound (T90) and the fact that the rate of degradation for a first order reaction decreases 50% for every 10 °C drop in temperature. Determination of Distribution Coefficients. Partitioning of compounds between n-octanol and aqueous buffer was determined at pH 7.4 using 0.1 M phosphate buffer (shake-flask method). The initial concentration (C 1 ) of compound in buffer and the buffer concentration following extraction with n-octanol (C2) were determined by RP-HPLC analysis against concentration standards for the specific compound. The distribution coefficient (DC) of a compound at a given pH was calculated using the equation DCpH = (C1 − C2)/C2. In Vitro Permeability and P-Glycoprotein Efflux. Permeability and transport studies were conducted and the data analyzed at Absorption Systems, Exton, Pennsylvania, using methods previously described.10 Briefly, MDCK(MDR) monolayers were grown to confluence on collagen-coated, microporous, polycarbonate membranes in 12-well Costar Transwell plates. To ensure monolayer integrity, the trans-epithelial electrical resistance (TEER) was measured. Only cell monolayers with TEER values >1900 Ω·cm2 were used. The permeability assay buffer was Hank’s Balanced Salt Solution containing 10 mM HEPES and 15 mM glucose at a pH of 7.0−7.2. Permeability through a cell-free (blank) membrane determined nonspecific binding and free diffusion of the test article through the device. Solution concentrations of the test articles were 10 μM in assay buffer. At each time point, 1 and 2 h, a 200-μL aliquot was taken from the receiver chamber and replaced with fresh assay buffer. Cells were dosed on the apical side [apical-to-basolateral, absorptive transport, (A-B)] or basolateral side [basolateral-to-apical, secretory transport, (B-A)] and incubated at 37 °C with 5% CO2 and 90% relative humidity. Each determination was performed in duplicate. Lucifer Yellow permeability was measured for each monolayer after the experiment to ensure that the cell monolayer integrity and viability were not compromised by the test article. Postexperiment Lucifer Yellow permeability in monolayers was 0.24 to 0.75 nm/s. To determine the transport of compounds in the absence of functional P-gp activity, the above experimental conditions were used but in the presence of the P-gp inhibitor cyclosporin A (CSA).13 Cells were preincubated for 30 min with the inhibitor (10 μM), then washed. During the permeation determination period, CSA (10 μM)

was present on both sides of the membrane. The Papp A-B determined in the presence of CSA was taken as an estimate of the permeability attributed to passive diffusion for the compound (Papp PD). Ex Vivo Determination of Ocular Tissue Permeability. These studies were conducted and the data analyzed at Absorption Systems, LP, Exton, PA using their standard methods. In brief, the corneal and palpebral conjunctival tissues were excised from male Dutch-belted pigmented rabbits (1.5−2.5 kg body weight, 3−3.5 months old) and mounted on a Harvard vertical diffusion apparatus with a diffusion area of 0.64 cm2. Preheated (37 °C), pH 7.4, GBR buffer was added to the mucosal (1.5 mL) and the serosal (1.5 mL) chambers. The diffusion apparatus was maintained at 37 °C throughout the entire transport experiment. Oxygenation and agitation were achieved by bubbling O2/ CO2 (95:5) through each chamber (at a rate of 5−6 bubbles per second). After the 30 min equilibration, blank GBR buffer in the mucosal (donor) chamber was withdrawn and replaced with GBR buffer containing 10 μM test compound. The transport experiments lasted 2 h and were performed in quadruplicate. Every 60 min, 0.2 mL samples were collected from the serosal (receiver) chamber and replenished with 0.2 mL blank GBR buffer, except at the last time point; at the end of the experiment, samples were also collected from the mucosal (donor) chambers for mass balance determination. After the transport experiment, tissue integrity was assessed by measuring the permeation of low permeable marker compounds atenolol and Lucifer yellow across the tissue. Donor chambers were replaced with GBR buffer containing 100 μM atenolol and 500 μM Lucifer yellow, and receiver chambers were replaced with fresh blank GBR buffer. After incubation for 30 min, samples were collected from both chambers for analysis. Test compounds and atenolol were analyzed by LC-MS/MS methods, and Lucifer yellow was determined by fluorescence detection. In Vitro Binding Assays. Serotonin Human 5-HT1A Receptor Binding. The procedure was previously described.9 In brief, the binding of [3H]-8-OH-DPAT (0.25 nM final) to Chinese hamster ovary cell membranes expressing the recombinant human 5-HT1A receptor was performed in 50 mM Tris-HCl buffer (pH 7.4) in a total volume of 0.5 mL for 1 h at 27 °C. Unlabeled 8-OH-DPAT (10 μM final) was used to define the nonspecific binding. The assays were terminated by rapid vacuum filtration and the samples counted on a scintillation counter. The data were analyzed using a nonlinear, iterative curve-fitting computer program. Serotonin Rat 5-HT2A Receptor Binding. The procedure was previously described.7 In brief, the relative affinities of compounds at the 5-HT2A receptor were determined by measuring their ability to compete for the binding of the agonist radioligand [125I]-DOI to rat brain 5-HT2A receptors. Aliquots of post-mortem rat cerebral cortex homogenates (400 μL) dispersed in 50 mM Tris-HCl buffer (pH 7.4) were incubated with [125I]-DOI (80 pM final) in the absence or presence of methiothepin (10 μM final) to define total and nonspecific binding, respectively, in a total volume of 0.5 mL. The assay mixture was incubated for 1 h at 23 °C in polypropylene tubes and the assays terminated by rapid vacuum filtration over Whatman GF/B glass fiber filters previously soaked in 0.3% polyethylenimine using ice-cold buffer. The samples were counted on a β-scintillation counter and the data analyzed using a nonlinear, iterative curve-fitting computer program. Cloned Human Serotonin 5-HT2 Receptor Binding. Binding affinity of compounds at the cloned human 5-HT2A, 5-HT2B, and 5HT2C receptors expressed in Chinese hamster ovary cells using the agonist [125I]-DOI (0.2 nM; 15 min at 37 °C) as the radioligand for each receptor was determined and reported as Ki values. These studies were conducted and the data analyzed at Cerep, Poitiers (France) using standard radioligand binding techniques as described above. Determination of Binding at Other Receptors and Cellular Elements. Binding assays for 5-HT1B, 5-HT1D, 5-HT3, 5-HT4, and 5HT6 serotonergic receptors and α1A, α1B, and α2B adrenergic receptors, and other receptor, enzyme, ion channel profiling were conducted at NovaScreen Biosciences (Hanover, MD) using their standard documented screening protocols. M

DOI: 10.1021/acs.jmedchem.5b00857 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Adrenergic Cloned Human α2A and α2C Receptor Binding. Membranes from Sf9 cells expressing the cloned human α2A and α2C receptor (Biosignal, Montreal Canada) were diluted to 32 μg/mL and 48 μg/mL protein, respectively, in 75 mM Tris-HCl containing 12.5 mM MgCl2 and 2 mM EDTA (pH 7.4). The membranes were resuspended using a Branson Sonifier 450 (Branson Ultrasonics Corp., Danbury, CT) (80% of their responses on the DOM-appropriate liver following administration of this drug dose. Administration of lower DOM doses resulted in the animals making fewer responses on the DOM-appropriate lever (ED50 = 0.3 mg/kg, 95% CL = 0.2−0.6 mg/kg; 1.25 μmol/kg). The response rate of the animals following the administration of different DOM doses was statistically similar (p > 0.05) to their response rate following the administration of the saline vehicle (13.1 ± 2.1

responses/min). The test compounds were examined in the DOMtrained animals as outlined above. Animal studies were conducted under an approved Institutional (VCU) Animal Care and Use Committee protocol.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00857. Additional ligand binding data for 13; elemental analysis data; thermal ellipsoids for 13; unit cell packing diagram; crystal data and structure refinement for 13 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 817-975-9199. E-mail: [email protected]. Present Addresses

(N.A.S.) Santen, Inc., Emeryville, CA. (B.S.S.) OMM Scientific, Inc., Dallas, TX. (C.R.K.) DiaTech Oncology, Franklin, TN. (W.F.H.) Xenanova Consulting, Dallas, TX. (M.R.H.) Novartis Institutes of Biomedical Research, Cambridge, MA. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Gary Williams, Parvanaeh Katoli, Colene Drance, and Shouxi Xu of our Molecular Pharmacology Unit; Daniel Scott, Tony Wallace, and Lanaya Wood of our In Vivo Pharmacology Unit; and Wayne Schneider of our Preformulation Unit for their expert technical assistance. We also thank Dr. John Liao of our Physical Characterization Unit for conducting the DC and stability studies and Dr. Vincent M. Lynch, Department of Chemistry and Biochemistry, University of Texas, Austin, for conducting the X-ray studies.

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ABBREVIATIONS USED DOM, 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane; 5MeO-DMT, 5-methoxy-N,N-dimethyltryptamine; IOP, intraocular pressure; NHP, nonhuman primate; TM, trabecular meshwork

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REFERENCES

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DOI: 10.1021/acs.jmedchem.5b00857 J. Med. Chem. XXXX, XXX, XXX−XXX